Lethal toxin is a major virulence factor of Bacillus anthracis. Lethal toxin (LT) is comprised of two proteins, protective antigen (PA) and lethal factor (LF). Macrophages serve as the first line of defense against anthrax infection. There is a striking difference in susceptibility of mouse strains and the primary macrophages or macrophage cell lines derived from those strains to lethal, toxin-mediated effects. Certain mouse strains and their macrophages are sensitive to LT whereas other mouse strains and their macrophages are highly resistant to LT. The underlying mechanism of resistance remains unknown and is a subject of great interest in the anthrax field due to the potential usefulness of this information to development of therapeutics. The primary goal of this project is to investigate mechanisms of resistance to LT in macrophages. To address this goal, three specific aims are proposed: (1) Investigation of alterations in cellular trafficking and localization using immunofluorescence/confocal microscopy to examine localization of LF in sensitive versus resistant macrophages, (2) Investigation of the role of potential LF intracellular substrates in resistant macrophages, and (3) Analysis of differential microarray analyses to evaluate gene expression in LT-sensitive versus resistant macrophages with a focus on evaluating cytokine expression, MAPK kinase effector molecule expression, and expression of Kif1C, a recently described murine gene product associated with resistance to LT. Successful completion of these studies will yield a fundamental understanding of mechanisms of macrophage resistance to LT.